RESUMEN
BACKGROUND: The main aim of this research was to explore the role and mechanism of Andrographolide (Andro) in controlling non-small cell lung cancer (NSCLC) cell proliferation. METHODS: Human NSCLC H1975 cells were treated with Andro (0-20 µM) for 4-72 h. B-cell leukemia/lymphoma 2 (Bcl-2)-antagonist/killer (Bak)-small interfering RNA (siRNA) (Bak-siRNA) and fructose-1,6-bisphosphatase (FBP1)-siRNA were transfected into H1975 cells to inhibit the endogenic Bak and FBP1 expression, respectively, and their expressions were detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting (WB). Cellular proliferation ability was determined through various assessments, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and cell counting kit-8 (CCK-8) assays. Cell apoptosis ability was measured using flow cytometry. Pro-apoptotic-related proteins (cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3) and mitochondrial apoptosis pathway proteins [Bcl2-associated X (Bax), Bak, Bcl-2, and cytochrome C (cyto C)] were assessed by WB. Aerobic glycolysis-associated genes [pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporter 1 (GLUT1)] and gluconeogenesis genes [phosphoenolpyruvate carboxykinase 1 (PEPCK1), fructose-1,6-bisphosphatase 1 (FBP1), and phosphofructokinase (PFK)] were measured by qRT-PCR. The mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1) assay was used for the measurement of mitochondrial membrane potential (ΔΨm). Additionally, glycolytic metabolism, lactate production, and adenosine triphosphate (ATP) synthesis were also analyzed. RESULTS: Andro inhibited human NSCLC cellular proliferation and induced apoptosis in a dose-time or dose-dependent manner via activation of the mitochondrial apoptosis pathway. Andro inhibited glycolysis, promoted the gluconeogenesis pathway, and increased the levels of cleaved caspase 9, cleaved caspase 8, cleaved caspase 3, Bax, Bak, PEPCK1, FBP1, and PFK, and decreased the levels of Bcl-2, PKM2, LDHA, and GLUT1. Moreover, it also decreased the ΔΨm and facilitated the release of cyto C from mitochondria into the cytoplasm. Furthermore, Andro enhanced the mitochondrial translocation of Bak, glucose uptake, lactate release, and intracellular ATP synthesis. Suppression of endogenic Bak and FBP1 expression significantly reduced the effects of Andro in H1975 cells. CONCLUSIONS: Andro represses NSCLC cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming glucose metabolism.
RESUMEN
BACKGROUND Thyroid cancer (TC) is one of the most prevalent endocrine malignancies and there may be many unclarified molecular events and gene types involved in TC. The objective of this study was to assess the clinical implications and potential mechanisms of serum response factor (SRF) in TC. MATERIAL AND METHODS RNA-sequencing and gene chip data with TC expression were collected from The Cancer Genome Atlas/Genotype-Tissue Expression, Gene Expression Omnibus, ArrayExpress, Sequence Read Archive, and Oncomine. SRF expression of all TC and adjacent non-cancerous tissue were calculated using the t test, STATA, and Meta-DiSc. The related pathways of the potential SRF target genes and target miRNAs were explored. Dual-luciferase reporter assay was performed to validate the association between SRF and its putative miRNA. RESULTS One RNA-sequencing and 15 gene chips were collected, and the pooled standardized mean difference of SRF was -1.00. Furthermore, the area under the curve of sROC of SRF in TC was 0.8251, indicating a dramatic decreased expression of SRF in TC tissues based on 1118 cases. The intersection of differentially expressed genes in TC, SRF co-expressed genes, and SRF potential target genes achieved from Cistrome Cancer led to 169 overlapped genes. miR-330-5p was predicted to target SRF, which was further confirmed by dual-luciferase reporter assay. CONCLUSIONS The reduction of SRF appears to play a crucial role in the origin of TC. These properties are accomplished by the target genes of SRF, as a transcription factor, or by the axes with the associated miRNAs.
